Estrogen has been shown to influence mammary gland growth, differentation and function during an animal's normal reproductive cycle. However, little is known concerning the cellular mechanism by which estrogen mediates these processes. We propose to develokp an immunohistochemical assay for the detection of individual mammary cells which contain estrogen receptor (estrophilin), an intracellular protein responsible for the mediation of estorgen action. To this end, we will produce specific polyclonal and monoclonal antibodies raised against estrophilin collected and purified from rat or rabbit mammary cells during lactation. The antibodies will be produced by the technique of Kohler and Milstein (1) as modified by McKearn et al. (2) in which spleen cells from immunized rats will be fused with mouse myeloma cells. This technique already has been successful in our laboratory (see appendix). The various hybrids will be screened for the production of antibody and cloned by limiting dilution. The antibodies from the mono-colonies will be purified by immunoadsorption techniques. (3) The appropriate antibodies will be 1) conjugated with a fluorescence-derivative, incubated with tissue, and examined under a high resolution microscope utilizing a computerized image intensifier; or 2) used in an "indirect sandwich technique" as described by Sternberger (4,5) and examined under an electron microscope for the prescise intracellular location of these receptor sites. Our initial objective is to establish and validate the immunohistochemical assay through a comparison with biochemically-derived binding data. Our long-term goal is to better understand the complex manner in which numerous hormones (insulin, GH, prolactin, oxytocin, progesterone...) modulate the various biological responses observed in the mammary gland during the reproductive cycle. In this project, we will initiate such endeavors by examining the mechanism of estrogen action. From this study we should be able to gain knowledge pertinent to the following objectives: 1) the type of mammary cells which contain estrophilin including mammary glands from immature animals; 2) the intracellular location of estrophilin in individual mammary cells; 3) the presence of a pre-estrophilin; and 4) possible alterations in #1-3 during the normal reproductrive cycle.